Mol. Hum. Reprod. Advance Access originally published online on July 28, 2005
Molecular Human Reproduction 2005 11(8):561-566; doi:10.1093/molehr/gah199
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Growth dynamics of human leiomyoma cells and inhibitory effects of the peroxisome proliferator-activated receptor-
ligand, pioglitazone
Department of Obstetrics and Gynecology, National University of Singapore, Singapore
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, National University Hospital, Lower Kent Ridge Road, 119074, Singapore. E-mail: obgyel{at}nus.edu.sg
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Abstract |
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Uterine leiomyomas (fibroids) are the most frequent tumour of the female reproductive tract and are the primary cause of hysterectomies in women worldwide. Effective treatment options are few. In a search for alternative treatments, we have established primary cultures of human leiomyoma cells and adjacent myometrial tissues, and documented their growth dynamics in response to estradiol (E2) and pioglitazone (PIO), a peroxisome proliferation-activated receptor-
(PPAR) ligand, currently in clinical use for type II diabetes mellitus. Human uterine primary cell cultures display morphology and desmin content consistent with their smooth muscle origin. Surprisingly, leiomyoma cells exhibited slower proliferation patterns relative to matched myometrial cells, both in the absence and presence of E2, suggesting that tumour genesis may not be because of increased growth potential but could be related to suppression of growth-inhibiting factors in vivo. PIO significantly inhibited the cell proliferation of both myometrial and leiomyoma cells in a dose-dependent manner. Our results suggest the possibility of using PPAR ligands, such as PIO, as therapeutic agents for the conservative management of uterine fibroids. Key words: diabetes/growth inhibition/leiomyoma/PPAR/pioglitazone
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Introduction |
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The smooth muscle layer of the uterus is the site of an extremely common neoplasm leiomyoma, more commonly known as fibroids. These tumours may occur in up to 30–70% of adult women depending on ethnicity (Stewart, 2001
). Although non-malignant in nature, leiomyomas result in abnormal menstrual bleeding, anaemia, abdominal pain, urinary symptoms and infertility. Indeed uterine fibroids are the most commonly cited reason for hysterectomies in Singapore and the United States of America (Chan et al., 1993; Farquhar and Steiner, 2002). Despite the morbidity that they cause and their significant impact on women’s health, many of the basic aspects of fibroid pathogenesis and growth remain unknown. Although conservative treatment options are available, undesirable side effects remains a substantial problem. It is well established that the primary stimuli for leiomyoma growth are estrogens and gonadotropin-releasing hormone agonists (GnRH), which induce a hypoestrogenic milieu that can shrink leiomyomas. However, such therapy leads to severe menopausal symptoms and osteoporosis. Cessation of GnRH therapy brings about a rapid return of ovarian steroidogenesis and regrowth of the tumours. Various alternative minimally invasive procedures, including uterine–artery embolization, are being developed (Falcone and Bedaiwy, 2002). However, such conservative treatments are associated with post-procedure complications and are palliative rather than curative and carry the same risks of fibroid reformation after treatment (Marret et al., 2005). It remains a challenge to discover novel non-surgical treatments that do not merely ameliorate the symptoms of the disease but cure existing fibroids and/or prevent the initiation of fibroid growth.
The effects of estradiol (E2) on fibroid development have been reported to be augmented by a long list of growth factors (Nowak, 2001). Growth-promoting proteins such as platelet-derived growth factor, heparin-binding epidermal growth factor, hepatoma-derived growth factor, basic fibroblast growth factor, transforming growth factor-beta and insulin-like growth factors and their associated signalling pathways and gene products have been implicated in the pathogenesis of leiomyomas (Gao et al., 2001; Lee and Nowak, 2001; Chegini et al., 2002; Xu et al., 2003). On the other hand, tumourigenesis have been related to the suppression of growth-inhibiting factors such as the tumour suppressor p53 (Gao et al., 2002; Shime et al., 2002), the apoptotic-related molecules Fas and Fas ligand, caspases, BCL-2 (Huang et al., 2002) and tumour necrosis factor alpha (Kurachi et al., 2001). Because E2 is the main stimulus for fibroid growth, we reasoned that factors that, directly or indirectly, oppose E2 action may be good candidates as therapeutic agents (Wang and Kilgore, 2002; Houston et al., 2003; Qin et al., 2003). In this regard, activators of the peroxisome proliferation-activated receptor (PPAR) family are attractive candidates. Both estrogens and PPAR agonists act through specific estrogen receptor (ER) and PPAR, members of the steroid/nuclear receptor family of transcription factors that regulate critical genes essential for sexual and embryonic development (McKenna and O’Malley, 2002). The thiazolidinedione class of PPAR ligands conveys anti-proliferative signals on several cell types, including vascular smooth muscle, pancreatic cancer, gastric cancer, renal cell carcinoma, colon cancer and breast cancer cells (Bruemmer and Law, 2003; Michalik et al., 2004). Ligands of PPAR have been reported to antagonize estrogen action (Dang et al., 2003) and preliminary reports indicate that the PPAR ligands 15-deoxy-
12,14-prostaglandin J2 and thiazolidinediones have inhibitory effects on the proliferation of leiomyoma cells (Houston et al., 2003; Young et al., 2004). If PPAR agonists such as pioglitazone (PIO) can be demonstrated to have anti-proliferative effects on human leiomyoma cells, they might have potential for human application as they have been used safely as antidiabetic agents for long periods. To examine the validity of this line of reasoning, we have established primary cultures of matched myometrial and fibroid cells from patients and determined their growth patterns. We used this model to study the effects of the PPAR ligand, PIO, on leiomyoma cell proliferation.
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Subjects and methods |
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Tissues
Uterine leiomyoma and matching normal myometrial tissues were obtained from premenopausal women who underwent elective abdominal hysterectomy for symptomatic uterine fibroids at the National University Hospital, Singapore. Cases with endometrial cancer were excluded. The menstrual cycle was recorded in those with regular cycles and was classified into follicular or luteal phases. Informed consent for the use of uterine tissues was obtained from each patient before surgery. The National University Hospital Institutional Review Board approved the protocol for tissue collection and their use in culture experiments.
Cell culture
Uterine leiomyoma and normal adjacent myometrial tissues were dissected from endometrial cell layers, cut into small pieces and digested in 2mg/ml collagenase type I (Invitrogen, Carlsbad, CA, USA) at 37°C for 3–5 h. Cells were collected by centrifugation at 460 x g for 5 min and washed three times with Dulbecco’s modified Eagle’s medium (DMEM) containing 1% antibiotic–antimycotic solution (Sigma, St. Louis, MO, USA). The isolated cells were plated at approximately 5 x 105 cells/dish in 25 cm2 culture dishes and subcultured at 37°C in a humidified atmosphere of 5% CO2–95% air in DMEM (Sigma) supplemented with 10% fetal calf serum (FCS, vol/vol; Biological Industries, Kibbutz Beit Haemek, Israel), 2 mM L-glutamine, 0.1 mM non-essential amino acid and 1 mM sodium pyruvate (Biological Industries, Kibbutz Beit Haemek, Israel). Cell culture media were changed every 3 days and cells passaged when they reached confluency. For all experiments, cells between passage three and nine were used and the cell culture medium was changed to DMEM supplemented with 10% charcoal-treated FCS 24 h before initiation of treatment. Total number of viable cells was determined using trypan blue (Sigma) exclusion assay and cell numbers following treatments counted manually with a hemocytometer after appropriate dilution.
MTT assay
In this assay the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) is converted to a coloured formazan product by mitochondrial dehydrogenase in living cells. Cells were seeded into 96-well microtiter plates at a density of 2 x 103/well. Twenty-four hours after inoculation, cells were exposed to vehicle or PIO (Takeda Chemical Industries Ltd, Osaka, Honshu, Japan) at final concentrations of 3, 10, 30 and 100 µM. On the designated day, MTT (Duchefa, Haarlem, Netherlands) reagent was added to each well to a final concentration of 0.5 mg/ml. After incubation at 37°C for 5 h, the formazan product was dissolved by addition of 200 µl DMSO (JT Baker, Phillipsburg, NJ, USA) and absorbance of the lysate was measured at 550 nm using a microplate reader (SLT Rainbow, SLT Labinstruments, Salzburg, Austria) with a reference wavelength of 650 nm. At least two independent experiments were performed in triplicate for each data point shown.
Immunohistochemical staining
Characterization of the cultured cells was performed using avidin/biotin immunoperoxidase method as previously described (Loy et al., 2003). Mouse monoclonal antibody to human desmin (Dako A/S, Glostrup, Copenhagen, Denmark) was used at a dilution of 1:50 as the primary antibody. The sections were subsequently washed in phosphate-buffered saline (PBS), incubated for 1 h with a second antibody [biotin-labelled anti-mouse immunoglobulin G (IgG); 1/200 in blocking solution; Dako A/S] and washed again in PBS. Sections were then incubated for 1 h with a tertiary antibody (Extravidin-TRITC, Sigma), diluted 1:200 in blocking solution, washed in PBS and mounted using FluorSave Reagent (Calbiochem, La Jolla, CA, USA).
Protein extraction and western immunoblotting
At the termination of cultures, cells were scraped off the plates and collected for immunoblotting as previously described (Wang et al., 2001). Blots were decorated with anti-cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a specific ER
mouse monoclonal antibody raised against full-length ER (Novocastra laboratories, Newcastle-upon-Tyne, UK). The antigen–antibody complexes were detected with the secondary antibody using the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Uppsala, Sweden). Membranes were visualized by exposure to CL-XPosure film (Pierce, Rockford, IL,USA). The images were then scanned and quantified with Scion Image software.
Statistical analysis
The experiments were performed in triplicates and repeated at least two times. The significance of differences was assessed by the Student’s t-test or the paired t-test where appropriate. Differences were considered to be significant when P < 0.05.
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Results |
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Six matched fibroids and adjacent myometrial tissue were collected from patients between October 2003 and May 2004. Cells from one patient, aged 52, displayed retarded growth patterns and inconsistent desmin staining and was excluded from this analysis. The remaining tissues from five subjects were used in this report. Their average age was 47.2 ± 1.4 (±SEM, range 44–52) years and average parity was 2.6 ± 0.8 (±SEM, range 0–5). All were of Chinese ethnic origin.
Growth dynamics of myometrial and leiomyoma cells in vitro
Cells isolated from human myometrial and leiomyoma tissues displayed morphology that was spindle-like and elongated and grew in a whorling pattern, characteristics typical of smooth muscle cells (Figure 1, upper panels). The smooth muscle nature of these uterine cells cultured ex vivo was further confirmed by immunoflourescence staining with antibodies directed against desmin, a muscle-specific intermediate filament that is not present in fibroblasts or vascular smooth muscles (Figure 1, lower panels). These characteristics were retained during their propagation under the cell culture conditions used in this study. To determine their ex vivo growth characteristics, myometrial and leiomyoma cell proliferation was measured after 3 and 6 days of propagation in complete DMEM growth media. Menstrual phase had no observable effect on cell proliferation rates, although differences might not be apparent because of the small numbers studied. Unexpectedly, the adjacent myometrial cells grew more rapidly compared to their matched leiomyoma cells, with doubling time of approximately 2 and 4 days, respectively (Figure 2A). This behaviour was consistently observed in the uterine cells isolated from different patients regardless of menstrual phase. To determine whether the difference in growth rates can be recapitulated by differential accumulation of key cell cycle proteins markers such as cyclin D1, total protein extracts of exponentially growing myometrial and leiomyoma cells were analyzed by western blotting to detect the expression of cyclin D1 over a 24 h period. The results showed that a steady time-dependent rise in cyclin D1 levels was observed for the myometrial cells (Figure 2B). In contrast, little change was observed in the expression levels of cyclin D1 in leiomyoma cells during the same period suggesting a reduced G1 to S progression in these cells. Because the in vivo growth of the myometrium is dependent on the ovarian sex steroid hormone estrogen, we tested if the primary cell cultures remain responsive to estrogen stimulation. Figure 2C and D demonstrated that proliferation of both myometrial and leiomyoma cells were stimulated by addition of 17ß-E2 compared to vehicle controls. In addition, both cell types continue to express the ER throughout the duration of cell propagation (Figure 2E) indicating that the estrogen-dependent growth signalling pathway was retained in these cell cultures. It is of interest to note that ER protein was present in greater amounts in myometrial cells, perhaps contributing to its greater growth potential compared to leiomyoma cells in vitro.
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Inhibition of leiomyoma cell proliferation by the PPAR agonist, pioglitazone
To determine whether the PPAR ligand, PIO, had any anti-proliferative effects on patient-derived leiomyoma cells, the cells were treated with vehicle or increasing concentrations of PIO (3–100 µM) and cell growth was measured after 6 days of incubation. The results showed that PIO inhibited leiomyoma cell numbers in a dose-dependent manner compared to vehicle alone (Figure 3A). The anti-proliferative effect of PIO was potent and >50% inhibition was achieved with 30 µM PIO (P < 0.001). To rule out the possibility that potential confounding effects of PIO on mitochondria size, shape or function may influence the MTT cell proliferation assay, physical cell numbers following treatments were counted manually with a hemocytometer in a repeat experiment. Consistent with the MTT-based assays, the direct cell count method also showed a dose-dependent reduction in cell numbers when leiomyoma cells were treated with increasing doses of PIO (Figure 3B). In addition, there was no visible increase in the number of dead cells floating in the growth medium, irrespective of the presence or absence of PIO.
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Effects of pioglitazone on myometrial and leiomyoma cell proliferation
To determine the anti-proliferative efficacy of PIO within the fibroid patient population, we examined its effects on cells derived from five unrelated subjects. The combined results indicated that PIO significantly inhibited the growth of both myometrial and leiomyoma cells in a dose-dependent manner (Figure 4A and B). Growth inhibition was noticeable from day 3 onwards although vehicle-treated cells did not display any signs of growth retardation and continued to proliferate under the experimental conditions employed. Although the response of the myometrial cells to PIO treatment is very similar to that of the leiomyoma cells, the latter did show significant inhibition of cell numbers to 10 µM PIO at day 6 compared to the higher dose of 30 µM PIO for myometrial cells, thus hinting to a possibility that subtle differences may exist in the way both cell types respond to the anti-proliferative effects of PIO. The presence of E2 did not prevent the inhibitory effects of PIO (Figure 5A). On the other hand, the selective PPAR antagonist, GW6992 was able to reverse to some extent the suppressive effects of PIO (Figure 5B, PIO vs. PIO + GW), indicating that these effects may be partially effected through PPAR-dependent pathways.
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Discussion |
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Contrary to expectations, this study indicates that ex vivo cultures of normal myometrial cells grew with a shorter doubling time (2 days) compared with autologous cells derived from leiomyomas (4 days). This differential growth was observed both in the absence and presence of E2. Accumulation of cyclin D1 proteins during myometrial cell propagation was discernible over a 24 h period, whereas leiomyoma cyclin D1 levels remained indifferent, consistent with the notion that myometrial cells proliferate faster. The higher proliferative activity displayed by myometrial cell cultures is counter-intuitive and is in contrast to reports that leiomyomas have elevated mitotic indices and proliferating cell nuclear antigen expression compared with matched myometrial samples (Dixon et al., 2002
). Although this finding is under-appreciated, it is not totally uncorroborated. Carney et al. (2002) reported that the doubling time of myometrial cells was once every 2 days compared to once every 10 days for leiomyoma cells. In another study, four of eight matched myometrial samples displayed shorter doubling times compared to leiomyoma cells, the rest showing no apparent difference (Mason et al., 2003). Furthermore, tumour size was found to be independent of the proliferative activity of individual leiomyomas in the same patient, suggesting that factors other than proliferative capacity regulate tumour formation (Dixon et al., 2002). In combination these reports suggest the hypothesis that increased proliferative potential may not be the main mechanism for leiomyoma development, but rather it may be defective ‘braking’ or apoptotic signals, operative in vivo but not in vitro, that allow continued overgrowth of fibroid tissues.
In this study, we demonstrate that PIO can dose-dependently inhibit the proliferation of leiomyoma cells and supports the contention that thiazolidinediones may have therapeutic potential as non-hormonal agent for leiomyomas (Young et al., 2004). Our data is consistent with the growing appreciation of the growth inhibitory effects of PPAR ligands on various tumour cells including breast, pancreatic, gastric, liver, colon renal and vascular smooth muscle cells (Bruemmer and Law, 2003; Michalik et al., 2004). Inhibitory mechanism of PPAR ligands has not been clearly understood and evidence suggests that their modes of action may be PPAR ligand subtype dependent and cell type specific. For example, PPAR ligands rosiglitazone and ciglitazone were shown to inhibit vascular smooth muscle cell growth independently of the cyclin kinase inhibitors p21 and p27 (Hupfeld and Weiss, 2001). In contrast, troglitazone induces growth arrest in hepatocarcinoma cells by inhibiting proteasome mediated degradation of p27 (Motomura et al., 2004), which differs from the 15-deoxy-12,14-prostaglandin J2-induced proteasome-dependent degradation of cyclin D1 to inhibit growth in breast cancer cells (Qin et al., 2003). Apoptotic mechanisms have also been implicated in the inhibitory effects of PPAR ligands (Yin et al., 2004). Inhibition of human B lymphocytic leukaemia and T lymphocytes derived from multiple sclerosis patients by PIO treatment were accompanied by DNA condensation and down regulation of BCL-2 (Schmidt et al., 2004; Zang et al., 2004). The challenge for the future is to elucidate the mechanisms, whether through PPAR or otherwise, by which PIO exerts its ‘braking’ effect on the proliferation of normal and tumour cells. Indeed recent evidence indicates that survivor signals such as down-regulation of the tumour suppressor protein p53 by E2 (Gao et al., 2002), E2-enhanced secretion of the anti-apoptotic frizzled related protein 1 (Fukuhara et al., 2002) and/or IGF-1 induced expression of the anti-apoptosis BCL-2 protein (Gao et al., 2001) may be critical mechanisms for tumour genesis.
Because the inhibitory doses of PIO are within recommended therapeutic ranges for clinical treatment of type II diabetes (Budde et al., 2003; Diamant and Heine, 2003), our data has raised the possibility of initiating clinical trials to examine the anti-leiomyoma effects of PIO. Additional preclinical support is also provided by the finding that treatment with troglitazone in combination with E2 completely prevented the formation of abdominal leiomyomas in guinea pigs (Tsibris et al., 1999). PIO was able to prevent neointimal tissue proliferation and postangioplasty restenosis in coronary patients with diabetes (Takagi et al., 2003). The drug is well tolerated in healthy volunteers, the only adverse effect being induction of peripheral edema in about 18% of recipients (Zanchi et al., 2004). Because the risk of uterine fibroids increases with increasing body mass index (Shikora et al., 1991; Sato et al., 1998), it would be most interesting to determine whether obese women would benefit from the anti-proliferative and insulin-sensitizing effects of PIO administration.
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Acknowledgements |
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We thank Takeda Chemical Industries Ltd for supplying PIO. This work was supported by NHG/RPR-02005 from the National Health Group Cluster Research Fund, Singapore.
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Dedication |
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We dedicate this paper to our co-author Associate Professor F.K. Lim, a fine gynaecological surgeon and clinical researcher, who was tragically lost in Khao Lak, Thailand, during the tsunami of 26 December 2004.
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Submitted on November 29, 2004; revised on March 21, 2005, April 16, 2005, and May 30, 2005; accepted on June 5, 2005
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